Detection of tBid Oligomerization and Membrane Permeabilization by Graphene-Based Single-Molecule Surface-Induced Fluorescence Attenuation

The permeabilization of organelle membranes by BCL-2 family proteins is a pivotal step during the regulation of apoptosis; the underlying mechanisms remain unclear. Based on the fluorescence attenuation by graphene oxide, we developed a single-molecule imaging method termed surface-induced fluorescence attenuation (smSIFA), which enabled us to track both vertical and lateral kinetics of singly labeled BCL-2 family protein tBid during membrane permeabilization. We found that tBid monomers lie shallowly on the lipid bilayer, where they self-assemble to form oligomers. During the initiation phase of self-assembly, the two central hydrophobic helices (α₆ and α₇) of tBid insert halfway into the phospholipid core, while the other helices remain on the surface. In oligomerized tBid clusters, α₆ and α₇ prefer to float up, and the other helices may sink to the bottom of the membrane and cause the formation of transient two-dimensional, micelle-like pore structures, which are responsible for the permeabilization of membranes and the induction of apoptosis. Our results shed light on the understanding of tBid-induced apoptosis, and this nanotechnology-based smSIFA approach could be used to dissect the kinetic interaction between membrane protein and lipid bilayer at the single-molecule level with subnanometer precision.